Natural killer (NK) cells are a subset of large granular lymphocytes that act as cyto-toxic immune cells. NK cells can be identified by any number of known cell surface markers which vary between species (e.g., in humans CD56, CD16, NKp44, NKp46, and NKp30 are often used; in mice NK1.1, Ly49A-W, CD49b are often used). In an active state, NK cells are capable of killing certain autologous, allogeneic, and even xenogeneic tumor cells, virus-infected cells, certain bacteria (e.g., Salmonella typhi), and other target cells. NK cells appear to preferentially kill target cells that express little or no Major Histocompatibility Class I (“MHCI” or “MHC-I”) molecules on their surface. NK cells also kill target cells to which antibody molecules have attached, a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). In action against target cells, NK cells can release pore-forming proteins called perforins, proteolytic enzymes called granzymes, and cytokines/chemokines (e.g., TNFα, IFNγ, etc.) that directly lead to target cell apoptosis or lysis, or that regulate other immune responses. Upon activation, NK cells also may express Fas ligand (FasL), enabling these cells to induce apoptosis in cells that express Fas.
Sufficient NK cell activity and NK cell count typically are both necessary to mounting an adequate NK cell-mediated immune response. NK cells may be present in normal numbers in an individual, but if not activated these cells will be ineffective in performing vital immune system functions, such as eliminating abnormal cells. Decreased NK cell activity is linked to the development and progression of many diseases. For example, research has demonstrated that low NK cell activity causes greater susceptibility to diseases such as chronic fatigue syndrome (CFS), viral infections, and the development of cancers.
NK cell activity is regulated by NK cell activity-modulating receptors (“NKCAMRs” or simply “AMRs”), which may be specific for various ligands such as MHC-I molecules, MHC-I homologs, or other biological molecules expressed on target cells. NK cells in an individual typically present a number of activating and inhibitory receptors. The activity of NK cells is regulated by a balance of signals transduced through these activating and inhibitory receptors. Each type of NKCAMR is briefly discussed in turn below.
When somatic cells are either under stress, such in cancer progression or are infected, various molecules, such as MICA and MICB, are typically displayed on the surface of the stressed cells and normally displayed MHC-I molecules are “lost” from the cell surface (reduced in number and/or glycosylated such that they are not “seen” as “foreign” by the immune system). NKCAMRs are sensitive to these and other changes in potential NK target cells associated with cellular stress, disease, and disorder.
Most NKCAMRs appear to belong to one of two classes of proteins: the immunoglobulin (Ig)-like receptor superfamily (IgSF) or the C-type lectin-like receptor (CTLR) super family (see, e.g., Radaev and Sun, Annu. Rev. Biomol. Struct. 2003 32:93-114). However, other forms of NKCAMRs are known. The structures of a number of NKCAMRs have been elucidated (Id.). To better illustrate the invention, types of well understood NKCAMRs, with reference to particular examples thereof, are described here. However, several additional NKCAMRs are known besides those receptors explicitly described here (see, e.g., Farag et al., Expert Opin. Biol. Ther. 3(2):237-250) and the inventive compositions and methods described herein typically will also be applicable to these and other NKCAMRs.
NK Cell Activating Receptors (NKCARs)
Many NK cell activating receptors (NKCARs) belong to the Ig superfamily (IgSF) (such receptors also may be referred to as Ig-like receptors or “ILRs” herein). Activating ILR NK receptors (AILRs) include, e.g., CD2, CD16, CD69, DNAX accessory molecule-1 (DNAM-1), 2B4, NK1.1; killer immunoglobulin (Ig)-like activating receptors (KARs); ILTs/LIRs; and natural cytotoxicity receptors (NCRs) such as NKp44, NKp46, and NKp30. Several other NKCARs belong to the CLTR superfamily (e.g., NKRP-1, CD69; CD94/NKG2C and CD94/NKG2E heterodimers, NKG2D homodimer, and in mice, activating isoforms of Ly49 (such as Ly49A-D)). Still other NKCARs (e.g., LFA-1 and VLA-4) belong to the integrin protein superfamily and other activating receptors may have even other distinguishable structures. Many NKCARs possess extracellular domains that bind to MHC-I molecules, and cytoplasmic domains that are relatively short and lack the inhibitory (ITIM) signaling motifs characteristic of inhibitory NK receptors. The transmembrane domains of these receptors typically include a charged amino acid residue that facilitates their association with signal transduction-associated molecules such as CD3zeta, FcεRIγ, DAP12, and DAP10 (2B4, for example, appears to be an exception to this general rule), which contain short amino acid sequences termed an ‘immunoreceptor tyrosine-based activating motif’ (ITAMs) that propagate NK cell-activating signals. Receptor 2B4 contains 4 so-called ITSM motifs (Immunoreceptor Tyrosine-based Switch Motif) in its cytoplasmic tail; ITSM motifs can also be found in NKCARs CS1/CRACC and NTB-A. The cytoplasmic domains of 2B4 and SLAM contain two or more unique tyrosine-based motifs that resemble motifs presents in activating and inhibitory receptors and can recruit the SH2-domain containing proteins SHP-2 and SAP (SLAM-associated protein).
Stress-induced molecules, such as MIC-A, MIC-B, and ULBPs in humans, and Rae-1 and H-60 in mice, can serve as ligands for NKCARs, such as the NKG2D homodimer. Cellular carbohydrates, pathogenic antigens, and antibodies can also be NKCAR ligands. For example, NKR-P1 may bind to carbohydrate ligands and trigger NK cell activation, particularly against tumor cells which exhibit aberrant glycosylation patterns. Viral hemagglutinins may serve as ligands for natural cytotoxic receptors (NCRs), such as ILR NKCARs NKp30, NKp44, NKp46, and NKp80.
NKCARs can either directly transduce activating signals or can act in connection with adaptor molecules or other receptors (either in the context of a coordinated response between receptors that are sometimes singularly effective or in the context of coreceptor-receptor pairings). For example, NKCAR NCRs typically lack ITAMs and, accordingly, bind to adaptor molecules through a charged residue in their transmembrane domains (e.g., NKp30 associates with the CD3 zeta chain; NKp44 associates with DAP12 and/or KARAP; NKp46 is coupled to the CD3 zeta chain and FcRIγ chain), which are, in turn, able to recruit protein tyrosine kinases (PTKs) in order to propagate NK cell-activating signals. CD16, which is a NKCAR important to NK cell-mediated ADCC and cytokine production, associates with homodimers or heterodimers formed of CD3 zeta and/or gamma chains. NKG2D appears to play a complementary and/or synergistic role with NCRs and NKCARs in NK cell activation. Activation of NK cells against particular targets may require coordinated activation of multiple NKCARs or NCRs, or only action of a single receptor. Other triggering surface molecules including 2B4 and NKp80 appear to function as coreceptors for NK cell activation.
Activating isoforms of human KIRs (e.g., KIR2DS and KIR3DS) and murine Ly-49 proteins (e.g., Ly-49D and Ly-49H) are expressed by some NK cells. These molecules differ from their inhibitory counterparts (discussed below) by lacking inhibitory motifs (ITIMs) in their relatively shorter cytoplasmic domains and possessing a charged transmembrane region that associates with signal-transducing polypeptides, such as disulfide-linked dimers of DAP12,
NKCIRs NK Cell Inhibitory Receptors
ILR (IgSF) NK cell inhibitory receptors (NKCIRs) (I) include a number of different human KIRs, specific for HLA-A, -B, or -C allotypes (KIRs may recognize multiple alleles within a particular allotype—e.g., KIR2DL1 recognizes HLA-Cw-2, 4, and 6 allotypes). CTLR superfamily inhibitory receptors include members of the CD94/NKG2 protein family, which comprise receptors formed by lectin-like CD94 with various members of the NKG2 family, such as NKG2A, and recognize the nonclassical class I molecules HLA-E and Qa-1 in humans and mice, respectively, and the murine Ly49 molecules that recognize the classical class I MHC molecules in mice. In even further contrast, NKRP1A, Nkrp1f and Nkrp1d are inhibitory receptors whose ligands are not MHC-related but are CTLR family members expressed on various cell types, such as dendritic cells, macrophages, and lymphocytes.
MHC class I-specific NKCIRs include CTLR Ly-49 receptors (in mice); the IgSF receptors LIRs (Leukocyte Immunoglobulin-like Receptors, in humans), KIRs (e.g., p58 and p70 Killer-cell Immunoglobulin-like Receptors, in humans), and CTLR CD94/NKG2 receptors (in mice and humans). All MHC-I-specific NKCIRs appear to use a common inhibitory mechanism apparently involving phosphorylation of immunotyrosine inhibitory motifs (ITIMs) in their cytoplasmic domains in the course of MHC-I binding and recruitment of tyrosine phosphatases (e.g., SHP-1 and SHP-2) to the phosphorylated ITIMs, resulting in the inhibition of proximal protein tyrosine kinases (PTKs) involved in NK activation through NKCARs
Inhibitory CD94/NKG2 heterodimers formed from CTLR glycoproteins, comprise an ITIM-bearing NKG2 molecule (e.g., NKG2A) and bind to non-classical MHC-I molecules (e.g., HLA-E in humans and Qa-1 in mice).
Inhibitory Ly-49 receptors are murine type II membrane disulfide-linked homodimer CTLR glycoproteins, which bind to various MHC-I molecules and deliver typically dominant inhibitory (negative) signals to NK cells. Ly-49A, for example, binds to alpha1/alpha2 domains of MHC-I molecule H-2Dd, whereas Ly-49C binds H-2 Kb. Human NK cells appear to lack homologs of the murine Ly-49 receptors. Instead, human NK cells express KIRs, which are not found in mouse NK cells. Although human KIRs and mouse Ly-49 receptors lack structural homology, they are functionally orthologous: Both types of receptors bind to HLA class I on target cells, resulting in inhibition of NK-mediated cytotoxicity.
An important type of NKCIRs is the KIRs. Generally, KIRs are cell surface glycoproteins, comprising one to three extracellular immunoglobulin-like domains, which are expressed by some T cells as well as most human NK cells. A number of KIRs are well characterized (see, e.g., Carrington and Norman, The KIR Gene Cluster, May 28, 2003, available through the National Center for Biotechnology Information (NCBI) Worldwide Website: ncbi.nlm.nih.gov/books/bookres.fcgi/mono_003/ch1d1.pdf). Human KIRs include KIR2DL and KIR3DL (KIRs also may be referred to by various other names such as CD158e1, CD158k, CD158z, p58 KIR CD158e1 (p70), CD244, etc.) (see, e.g., US Patent Application 20040038894, Radaev et al., Annu. Rev. Biophys. Biomol. Struct., 32:93-114 (2003), Cerweknka et al., Nat. Rev. Immunol. 1:41-49 (2001); Farag et al., Expert Opin. Biol. Ther., 3(2):237-250 (2003); Biassoni et al., J. Cell. Mol. Med., 7(4):376-387 (2003); and Warren et al., British J. Haematology, 121:793-804 (2003), each of which being hereby incorporated in their entirety). The structure of a number of KIRs has been elucidated and reveals remarkable structural similarity between these proteins. See, e.g., Radaev et al., supra.
KIRs can be classified structurally as well as functionally. For example, most KIRs have either two Ig domains (58 kDa KIR2D KIRs), whereas others have three Ig domains (70 kDa KIR3D KIRs) (sometimes respectively referred to as p58 and p70 molecules). KIRs vary also in cytoplasmic tail length. Typically, KIRs with a relatively long cytoplasmic tail (L) deliver an inhibitory signal, whereas KR with a short cytoplasmic tail (S) can activate NK or T cell responses. Nomenclature for KIRs accordingly can be based upon the number of extracellular domains (KIR2D or KIR3D) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). Additional nomenclature information for KIRs is provided in the following Detailed Description of the Invention. Some members of the “KIR family” are NKCARs, or more particularly “KARs” (e.g., KIR2DS2 and KIR2DS4); they typically comprise one or more charged transmembrane residues (e.g., Lys) that associate with an adapter molecule having an immunostimulatory motif (ITAM) (e.g., DAP12). The intracytoplasmic portion of inhibitory KIRs typically comprises one or more ITIMs that recruit phosphatases. Inhibitory KIRs bind to alpha1/alpha2 domains of HLA molecules. Inhibitory KIRs do not appear to typically require adaptor-molecule association for activity. Unless otherwise stated, terms such as “KIR”, “KIRs”, and the like refer to NKCIR members of the “KIR family” and terms such as “KAR”, “KARs”, and the like refer to NKCAR members of the “KIR family.”
KIRs can bind MHC-I molecules (e.g., certain HLA class I allotypes), typically resulting in the transmission of a negative signal that counteracts, and may override stimulatory, activating signal(s) to the NK cell, thereby preventing the NK cell from killing the associated potential target cell (apparently via ITIM phosphorylation and tyrosine phosphatase (e.g., SH2-domain containing protein tyrosine phosphatases such as SHP-1 and SHP-2) recruitment, leading to PTK (e.g., Syk, TcR and/or ZAP70) dephosphorylation and/or LAT/PLC complex formation inhibition and associated disruption of ITAM cascade(s)). Because viruses often suppress class I MHC expression in cells they infect, such virus-infected cells become susceptible to killing by NK cells. Because cancer cells also often have reduced or no class I MHC expression, these cells, too, can become susceptible to killing by NK cells. Infected cells can also change the proteins bound in the MHC in terms of glycosylation. If this occurs, the MHC-I:protein complex the cell expresses will be altered. If NK-associated KIRs cannot bind to these “foreign” complexes, no inhibitory signal can be generated, and lysis will proceed.
All confirmed inhibitory KIRs appear to interact with different subsets of HLA/MHC antigens depending upon the KIR subtype. In humans, KIRs having two Ig domains (KIR2D) recognize HLA-C allotypes: KIR2DL2 (formerly designated p58.2) and the closely related gene product KIR2DL3 both recognize an epitope shared by group 1 HLA-C allotypes (Cw1, 3, 7, and 8), whereas KIR2DL1 (p58.1) recognizes an epitope shared by the reciprocal group 2 HLA-C allotypes (Cw2, 4, 5, and 6). The specificity of KIR2DL1 appears to be dictated by the presence of a Lys residue at position 80 of group 2 HLA-C alleles. KIR2DL2 and KIR2DL3 recognition appears to be dictated by the presence of an Asn residue at position 80. A substantial majority of HLA-C alleles have either an Asn or a Lys residue at position 80. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, a homodimer of molecules with three Ig domains, KIR3DL2 (p140), recognizes HLA-A3 and -A11.
Individual MHC-I-specific NK cell receptors of either type (activating or inhibitory) typically do not interact with all MHC class I molecules, but specifically bind to certain allotypes (proteins encoded by different variants of a single genetic locus). Also, an individual NK cell may express several different inhibitory and/or activating receptors which function independently of each other. For example, in humans the presence or absence of a given KIR is variable from one NK cell to another within a single individual. There also is relatively high level of polymorphism of KIRs in humans, with certain KIR molecules being present in some, but not all individuals. Although KIRs and other MHC-recognizing inhibitory receptors may be co-expressed by NK cells, in any given individual's NK repertoire there are typically cells that express a single KIR; accordingly, the corresponding NK cell activity in this latter type of NK cells is inhibited only by cells expressing a specific MHC-I allele group. In fact, recent estimates of the extent of KIR genotype diversity within the population suggest that <0.24% of unrelated individuals can expect to have identical genotypes. The most common Caucasian haplotype, the “A” haplotype (frequency of ˜47-59%), contains only one activating KIR gene (KIR2DS4) and six inhibitory KIR loci (KIR3DL3, -2DL3, -2DL1, -2DL4, -3DL1, and -3DL2). The remaining “B” haplotypes are very diverse and contain 2-5 activating KIR loci (including KIR2DS1, -2DS2, -2DS3, and -2DS5).
Antibodies against NK receptors, such as KIRs, have been previously described and there also has been at least some suggestion of combining anti-NK receptor antibodies, such as anti-KIR antibodies, with other anti-cancer agents in the prior art. For example, WO2004056392 describes anti-NKp30 and/or anti-NKp46 antibodies used in admixture with interleukin-2 (IL-2). WO2005009465 describes the combination of a therapeutic antibody (e.g, Rituxan) in combination with a compound that blocks an inhibitory receptor or stimulates an activating receptor of an NK cell (e.g., an anti-KIR mAb, such as the mAb DF200) in order to enhance the efficiency of the treatment with therapeutic antibodies in human subjects (see also US 20050037002). WO2005079766 also describes combinations of antibodies (e.g, anti-tissue factor antibodies) including anti-KIR antibodies for use in cancer therapies. WO2005003168 and WO2005003172 describe combinations of a number of anti-KIR antibodies with a variety of agents, including IL-2 and IL-21. WO2005037306 similarly describes combinations of IL-21, IL-21 derivatives, and IL-21 analogues in combination with anti-KIR antibodies.
The invention described herein relates to the treatment of cancer and pre-cancerous conditions wherein an antibody against a KIR is employed in combination with other cancer or cancer preventive treatments. Specific combinations described herein are new with respect to the prior art and in at least some instances associated with surprising properties, such as unexpected synergistic effects. The invention also relates to new methods and compositions useful for the treatment of viral infections, comprising a combination of an antibody against a KIR and an anti-viral medicament or therapeutic technique.